Recombinant Danio rerio Suppressor of tumorigenicity 7 protein homolog (st7)

What expression systems are optimal for recombinant Danio rerio ST7 production?

Multiple expression systems have been validated for zebrafish ST7 protein production. According to available data, recombinant Danio rerio ST7 can be effectively expressed in:

• Cell-free expression systems: Offering rapid production with reduced complexity for initial characterization studies .

• E. coli expression systems: Providing high yield though potentially lacking some post-translational modifications .

• Baculovirus expression: Suitable for producing ST7 with more complex folding requirements .

• Mammalian cell expression: Offering the most physiologically relevant post-translational modifications .

The selection depends on experimental requirements regarding protein yield, purity, and post-translational modifications. For structural studies requiring high purity, E. coli systems followed by SDS-PAGE validation to confirm ≥85% purity is recommended .

How can I verify the identity and purity of recombinant ST7 protein?

Verification protocols should include multiple complementary techniques:

• SDS-PAGE analysis: Should demonstrate ≥85% purity with the expected molecular weight band .

• Western blot analysis: Using validated antibodies such as rabbit anti-Danio rerio ST7 polyclonal antibodies for specific detection .

• Mass spectrometry: For peptide fingerprinting to confirm protein identity.

• Functional assays: To verify biological activity of the purified protein.

For optimal results, purified protein should be aliquoted, flash-frozen, and stored at -80°C to maintain stability and prevent freeze-thaw cycles.

What methodologies exist for creating ST7 knockout/knockdown zebrafish models?

Several approaches can be employed to generate ST7-deficient zebrafish models:

• Zinc Finger Nucleases (ZFNs): These can be designed to target specific sites within the st7 gene. The approach involves:

  • In silico analysis of the st7 cDNA sequence to identify potential zinc finger binding sites

  • Assembly of 4-finger ZFPs directed against distinct positions in the st7 locus

  • Validation of ZFP binding affinity using ELISA assays

  • Testing ZFN activity in a yeast-based system

  • Microinjection of validated ZFN mRNAs into 1-cell zebrafish embryos

  • Screening for mutations using PCR and restriction enzyme digestion

• CRISPR/Cas9 system: This more recent technique offers improved efficiency and specificity:

  • Design of guide RNAs targeting conserved exons of the st7 gene

  • Co-injection of guide RNAs and Cas9 mRNA into zebrafish embryos

  • Validation of mutations through sequencing and phenotypic analysis

Both approaches typically yield small insertions or deletions at the target site, which can disrupt gene function when appropriately positioned .

How can I assess potential off-target effects when using genome editing tools to modify the st7 gene?

When using genome editing tools such as ZFNs or CRISPR/Cas9 to target the st7 gene, off-target assessment is critical:

• In silico prediction:

  • Experimentally determine the DNA binding consensus for each nuclease

  • Search the zebrafish genome for sites showing highest similarity to the intended target

  • Identify the top 5 potential off-target loci for experimental validation

• Experimental validation:

  • Direct analysis of predicted off-target sites by PCR amplification and sequencing

  • Whole genome sequencing of selected mutant lines to identify unpredicted off-target effects

  • Complementation testing by crossing different mutant lines to confirm phenotypes are due to st7 disruption rather than off-target effects

• Phenotypic validation:

  • Perform rescue experiments by introducing wild-type st7 to confirm phenotype specificity

  • Compare multiple independently generated mutant lines to confirm consistent phenotypes

This multi-layered approach ensures that observed phenotypes are specifically related to st7 disruption rather than off-target effects .

What experimental approaches can elucidate ST7 function in zebrafish development and disease models?

To comprehensively investigate ST7 function in zebrafish, consider these methodological approaches:

• Spatiotemporal expression profiling:

  • Whole-mount in situ hybridization at different developmental stages

  • Transgenic reporter lines with fluorescent proteins under st7 promoter control

  • Quantitative PCR to measure expression levels across tissues and timepoints

• Loss-of-function studies:

  • Analysis of morphological phenotypes in st7 mutants/knockdowns

  • Histological examination of affected tissues

  • Behavioral assays to assess functional outcomes

• Disease model integration:

  • Challenge studies with pathogens to assess immune response changes in st7-deficient fish

  • Analysis of inflammatory markers and cytokine expression patterns

  • Evaluation of neutrophil and macrophage recruitment and function

These approaches should be applied within the developmental window where zebrafish rely exclusively on innate immune responses (up to 4-6 weeks post-fertilization), providing an opportunity to examine st7 function without the confounding effects of adaptive immunity .

How can interactions between ST7 and other proteins be identified in zebrafish?

Multiple complementary approaches can identify protein-protein interactions involving ST7:

• Co-immunoprecipitation (Co-IP):

  • Using anti-ST7 antibodies to pull down protein complexes from zebrafish tissue lysates

  • Mass spectrometry analysis of co-precipitated proteins

  • Validation of identified interactions using reverse Co-IP

• Proximity labeling approaches:

  • Generation of ST7-BioID or ST7-APEX2 fusion proteins

  • Expression in zebrafish through transgenesis or mRNA injection

  • Identification of proximal proteins through streptavidin pulldown and mass spectrometry

• Yeast two-hybrid screening:

  • Using ST7 as bait against a zebrafish cDNA library

  • Validation of positive interactions through secondary assays

  • In vivo confirmation in zebrafish embryos

Each method has distinct advantages and limitations, so combining multiple approaches provides the most robust identification of physiologically relevant interactions.

What techniques are most effective for measuring ST7 expression in zebrafish tissues?

For comprehensive ST7 expression analysis in zebrafish tissues:

• At the mRNA level:

  • Quantitative RT-PCR: Provides precise quantification across different tissues

  • In situ hybridization: Reveals spatial distribution within tissues

  • RNA-seq: Offers genome-wide context for st7 expression patterns

• At the protein level:

  • Western blotting: Using validated antibodies such as rabbit anti-Danio rerio ST7 for quantification

  • Immunohistochemistry: For spatial localization within tissue sections

  • ELISA: For quantitative measurement in tissue lysates

• For real-time visualization:

  • Transgenic reporter lines expressing fluorescent proteins under st7 promoter control

  • Time-lapse imaging during developmental processes

For developmental studies, stage-dependent expression analysis should be performed systematically from embryonic development through larval stages to maturity, as expression patterns may change significantly across development .

How should researchers analyze transcriptomic data to identify potential ST7-regulated pathways?

For transcriptomic analysis of ST7-regulated pathways:

• Experimental design recommendations:

  • Compare wild-type vs. st7 mutant/knockdown zebrafish at multiple developmental timepoints

  • Include biological replicates (minimum n=3 for each condition)

  • Consider tissue-specific RNA isolation for targeted analysis

• Bioinformatic analysis workflow:

  • Differential gene expression analysis using DESeq2 or similar tools

  • Pathway enrichment analysis using KEGG, GO, and Reactome databases

  • Upstream regulator prediction using tools like IPA or LISA

• Validation approach:

  • qRT-PCR confirmation of key differentially expressed genes

  • Protein-level validation of selected targets

  • Functional studies of identified pathways using pharmacological inhibitors or genetic approaches

This comprehensive approach allows for the identification of both direct and indirect targets of ST7 regulation, providing insights into its biological function.

What are the key challenges in characterizing post-translational modifications of recombinant ST7?

Several methodological challenges exist when characterizing post-translational modifications (PTMs) of recombinant ST7:

• Expression system selection:

  • E. coli systems lack eukaryotic PTM machinery

  • Insect cell systems may introduce different glycosylation patterns than native zebrafish

  • Mammalian expression systems provide more physiologically relevant PTMs but with lower yields

• Analytical approaches for PTM characterization:

  • Mass spectrometry protocols for phosphorylation site mapping

  • Glycan analysis through lectin affinity or specialized MS approaches

  • Site-directed mutagenesis to determine functional significance of identified PTMs

• Comparative analysis recommendations:

  • Parallel characterization of native ST7 purified from zebrafish tissues

  • Comparison across expression systems to identify critical modifications

  • Functional assays to determine the impact of specific PTMs

These challenges underscore the importance of selecting appropriate expression systems based on experimental requirements for studying ST7 function and structure.

How can researchers overcome solubility and stability issues with recombinant ST7 protein?

Recombinant proteins often present solubility and stability challenges. For ST7:

• Solubility enhancement strategies:

  • Fusion tags optimization (SUMO, MBP, or GST tags often improve solubility)

  • Expression temperature and induction optimization

  • Co-expression with molecular chaperones

  • Buffer screening using high-throughput approaches

• Stability assessment and improvement:

  • Differential scanning fluorimetry to identify stabilizing conditions

  • Addition of specific cofactors or binding partners

  • Identification of minimal stable domains through limited proteolysis

  • Storage condition optimization (cryoprotectants, pH, ionic strength)

• Quality control protocols:

  • Regular assessment of protein activity over time

  • Size-exclusion chromatography to monitor aggregation

  • Circular dichroism to evaluate secondary structure maintenance

These approaches should be systematically tested and optimized for the specific construct and expression system used for ST7 production.